1. Field of the Invention
This invention relates generally to gene amplification and specifically to a method for isolation of extrachromosomal amplified nucleic acid sequences.
2. Description of Related Art
Gene amplification in tumor cells results in the production of multiple copies of a genomic region. Amplification of oncogenes leads to the over expression of proteins participating in the transduction of growth-related signals and confers a growth advantage to tumor cells. Clinically, oncogene amplification is extremely common in human tumors and correlates with a poor prognosis for patients with ovarian cancer (HER-2/neu), breast cancer (c-myc, HER-2/neu), neuroblastoma (N-myc), or small cell lung cancer (c-myc) (Slamon, et al., Science 235:177, 1987; Slamon, et al., Science, 244:707, 1989; Seeger, et al., N. Engl. J. Med., 313:1111, 1985; Johnson, et al., J. Clin. Invest., 78:525, 1986). There is also evidence that amplification of drug resistant genes is associated with both in vitro and in vivo resistance of a patient""s tumor to an antineoplastic agent (Schimke, R., Cancer Res., 44:1735, 1984; Stark, G., Cancer Surv. 5:1, 1986; Trent, et al., J. Clin. Oncol., 2:8, 1984; Curt, et al., N. Engl. J. Med., 308:199, 1983).
Amplified genes have been localized to two types of cytogenetically distinguishable structures. These structures can be located on the chromosome, within homogeneously staining regions (HSRs), or they can be reside extrachromosomal either as submicroscopic elements called episomes or as larger structures called double minute chromosomes (Carroll, et al., Mol. Cell. Biol., 8:1525, 1988; Von Hoff, et al., J. Clin. Invest., 85:1887, 1990; Von Hoff, et al., Proc. Natl. Acad. Sci. USA, 85:4804, 1988). The occurrence of DMs in a malignant cell line was first described by Spriggs, et al. (Br Med J, 2:1431, 1962). DMs are paired, acentric fragments that segregate randomly at cell division and can be detected in the majority of primary tumors at biopsy (Benner, et al., Anticancer Drugs, 2:11, 1991; Gebhart, et al., Int. J. Cancer, 34:369, 1984). DMs tend to vary in size and also in number of DMs per cell.
Genes amplified on DMs can be lost spontaneously at each cell division or can be eliminated by treatment with hydroxyurea (HU) at concentrations that do not inhibit DNA synthesis or ribonucleotide reductase (Von Hoff, et al., Proc. Natl Acad Sci, USA, 89:8165, 1992; Von Hoff et al., Cancer Res., 51:6273, 1991). It appears that HU-treatment of cells containing DMs results in an increase in micronuclei formation, and the capture of the DMs within the micronuclei (Von Hoff, et al., Proc. Natl Acad Sci, USA, 89:8165, 1992; Von Hoff, et al., Proc. Am Assoc. Cancer Res., 33:359, 1992). By contrast, HSRs are not lost during cell division or by treatment with HU and represent a stable form of gene amplification. Elimination c-myc genes contained in DMs from a colon cancer cell line of neuroendocrine origin (COLO 320 DM) reduced its tumorigenicity in nude mice (Von Hoff, et al., Proc. Natl. Acad. Sci., USA, 89:8165, 1992). Studies have shown that treatment of HL60 cells with low concentrations of HU reduced the number of c-myc-containing DMs, which led to decreased c-myc expression and induction of differentiation (Echardt, et al., Proc. Natl. Acad. Sci., USA, 91:6674, 1994; Shimizu, et al., Cancer Res., 54:3561, 1994). The studies also showed that agents which eliminate extrachromosomal DNA also alter tumor phenotype.
Similarly, previous studies have shown that when the selective pressure of a drug is removed from mama cells that carry unstably amplified genes on extrachromosomal particles, the cells gradually lose those amplified genes (lose their DMS or episomes). Snapka and Varshavsky previously showed that HU could increase the rate of loss of unstably amplified dihydrofolate reductase (DHFR) genes from mouse cells (Proc. Natl. Acad. Sci., USA, 80:7533, 1983). Von Hoff, et al., showed a similar elimination of the multidrug resistance gene 1 (MDR1) gene in vinblastine resistant human squamous tumor cells, as well as carbamylphosphate synthetase, aspartate transcarbamylase, dihydro-orotase (CAD) genes from N-(phosphonacetyl)-L-aspartic acid (PALA) resistant Chinese hamster ovary cells, and DHFR genes from methotrexate resistant human squamous tumor cells (Cancer Research, 51:6273, 1991).
The persistence of DMs implies that these acentric elements express one or more genes that impart a growth or survival advantage to the cell. Identification of the expressed nucleic acid sequences contained in such DMs would provide a means for developing appropriate diagnostic, prognostic and therapeutic strategies.
The knowledge that gene amplification occurs in cancer cells provides an unparalleled opportunity for developing therapeutic approaches that are highly specific for tumor cells. The ability to eliminate amplified genes by removal or selective interference with their expression is enhanced by the determination of the identity of the amplified gene. The present invention provides a method for isolating and for identifying amplified genes which exist extrachromosomally in DMs within a cell.
The method of the invention allows isolation and molecular cloning of target nucleic acid sequences contained in extrachromosomal amplified loci. The identity of such nucleic acids or genes, whether previously known or unknown, provides a means for more accurate diagnosis and prognosis for a subject having a disorder such as a cancer. The identity of the genes also provides a method for monitoring the course of therapy for such a subject.